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WO1992012238A1 - Clonage et expression de transglutaminases tissulaires - Google Patents

Clonage et expression de transglutaminases tissulaires Download PDF

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WO1992012238A1
WO1992012238A1 PCT/US1991/009784 US9109784W WO9212238A1 WO 1992012238 A1 WO1992012238 A1 WO 1992012238A1 US 9109784 W US9109784 W US 9109784W WO 9212238 A1 WO9212238 A1 WO 9212238A1
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Peter J. A. Davies
Joseph P. Stein
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The Board Of Regents, University Of Texas System
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1025—Acyltransferases (2.3)
    • C12N9/104—Aminoacyltransferases (2.3.2)
    • C12N9/1044—Protein-glutamine gamma-glutamyltransferase (2.3.2.13), i.e. transglutaminase or factor XIII

Definitions

  • Transglutaminases are a group of calcium dependent enzymes that catalyze the crosslinking of proteins by promoting the formation of e-( ⁇ -glutaminyl)lysine isopeptide bonds between protein-bound glutamine and lysine residues. These enzymes are believed to be widely distributed in nature, as the crosslinks are found in both prokaryotic and eukaryotic cells. Although different transglutaminases appear to be very similar in substrate specificity, several distinct forms of the enzymes have been identified. See generally. Folk, Ann. Rev. Biochem. 49:517-531 (1980).
  • Transglutaminase-mediated protein crosslinking reactions have been implicated in both normal and pathological processes in mammalian cells and tissues.
  • the crosslink may act to maintain some forms of protein structure, such as in the terminal differentiation of epidermal cell layers and in other cellular architecture.
  • An intracellular transglutaminase known as epidermal or Type I transglutaminase has been isolated and cloned from rabbit epithelial cells (Floyd and Jetten, Mol. Cell. Biol. 9:4846-4851 (1989)), and a transglutaminase has been isolated and cloned from guinea pig liver cells (Ikura et al., Biochem. 27: 2898-2905 (1988)).
  • transglutaminases include hair follicle transglutaminase, keratinocyte transglutaminase, and prostate transglutaminase (Wilson et al., Fed. Proc. 38:1809 (1979)).
  • Lee et al.. Prep. Biochem. 16:321- 335 (1986) have described the purification of a transglutaminase from human erythrocytes.
  • These transglutaminases have been shown to be distinct from a plasma transglutaminase, Factor XIII, an enzyme whose primary function appears to be stabilizing fibrin clots.
  • Factor XIII has also been purified, cloned, and sequenced.
  • transglutaminases have been employed for crosslinking purposes in a variety of fields. Certain microbial transglutaminases have found use in food technology to add texture to processed foods, particularly fish and cheese. Others have been used in enzyme catalyzed fluorescent labeling of proteins, in the introduction of cleavable crosslinks, and in the solid phase reversible removal of specific proteins from biological systems. Factor XIII preparations have been proposed for a variety of therapeutic uses, such as the treatment of subarachnoid hemorrhage and inflammatory bowel disease.
  • a plasma derived Factor XIII is available as a fibrin sealant, but, as with most plasma-derived products, carries an inherent risk of viral contamination. Further, Factor XIII and certain other transglutaminases are zymogens, requiring some form of activation to become catalytically active. And, as each transglutaminase has a restricted range of substrates, their activity may be limited in certain applications. Accordingly, what is needed in the art are methods for producing by recombinant means human and murine transglutaminases, particularly those transglutaminases which do not require activation to become catalytically active. The present invention fulfills these and other related needs.
  • the present invention provides the ability to produce human and murine tissue transglutaminases and polypeptides or fragments thereof by recombinant means, preferably in cultured eukaryotic cells.
  • the expressed transglutaminase may or may not have the biological activity of the native enzyme, depending on the intended use. Accordingly, isolated and purified polynucleotides are described which code for the transglutaminases and fragments thereof, where the polynucleotides may be in the form of DNA, such as cDNA or genomic DNA, or RNA. Based on these sequences probes may be designed for hybridization to identify these and related genes or transcription products thereof which encode human and murine tissue transglutaminases.
  • the invention concerns DNA constructs which comprise a transcriptional promoter, a DNA sequence which encodes the transglutaminase or fragment thereof, and a transcriptional terminator, each operably linked for expression of the enzyme or enzyme fragment.
  • the constructs are preferably used to transform or transfect host cells, preferably eukaryotic cells, more preferably yeast or mammalian cells.
  • the expressed transglutaminase may be isolated from the cells by, for example, immunoaffinity purification.
  • Nucleic acid sequences which encode the transglutaminases of the invention and the recombinant transglutaminases themselves can also be used to develop compounds which can alter transglutaminase-associated apoptosis of a eukaryotic cell.
  • Compounds may be screened for agonistic or antagonistic effects on transglutaminase-mediated metabolism in the host cell.
  • Fig. 2 illustrates the sequencing strategy for the human cDNA insert in clone hTGl
  • Fig. 3 illustrates nucleotide sequences of human endothelial (SEQ. ID. No. 1) and mouse macrophage (SEQ. ID. No. 3) tissue transglutaminases and their predicted amino acid sequences (SEQ. ID. NO. 2 and SEQ. ID. NO. A , respectively), where the wavy lines indicate the amino acid sequence of the pentapeptide containing the active site cysteine residue, the nucleotide sequence corresponding to a putative polyadenylation signal in the mouse sequence is located at the position 3452- 3457, and the nucleotide sequence derived from mouse heart cDNA library has been underlined;
  • Fig. 4 illustrates the identification of human tissue transglutaminase mRNA by blot hybridization, analyzing 10 ⁇ g of mRNA from HUVEC;
  • Fig. 5 illustrates the identification of mouse transglutaminase mRNA by blot hybridization, analyzing 10 ⁇ g of mRNA from each of the following tissues: L, liver; S, spleen; K, kidney; T, testis; H, heart; Lu, lung; Tm, thymus; and B, brain.
  • Tissue transglutaminase (or transglutaminase II) is an enzyme that catalyzes the crosslinking of protein-bound glutamine and primary amines, such as lysine residues.
  • the present invention provides isolated nucleotide sequences of human tissue transglutaminase, thereby providing for the ultimate expression of human tissue transglutaminase polypeptides. Recombinant DNA expression systems provide convenient means for obtaining large quantities of human tissue transglutaminases in relatively pure form.
  • the invention also provides cloned nucleotide sequences of murine tissue transglutaminase.
  • the invention also provides recombinant human and murine tissue transglutaminase polypeptides and fragments thereof having transglutaminase activity.
  • polypeptides and fragments is meant to include sequences of amino acids up to entire proteins, which have at least about 85% homology, preferably at least 90%, and more preferably at least about 95% or more homology to the amino acid sequences of the murine or human sequences of the invention, as shown in Fig. 3 and SEQ. ID. Nos. 1-4.
  • the invention also includes those polypeptides having slight variations in amino acid sequences or other properties.
  • Such variations may arise naturally as allelic variations (e.g., due to genetic polymorphism) or may be produced by human intervention (e.g., by mutagenesis of cloned DNA sequences), such as induced point, deletion and insertion mutants.
  • Nucleic acid sequences encoding human tissue transglutaminase as described herein can be cloned from a variety of human cell sources that express the enzyme. Preferred sources include human umbilical vein endothelial cells and retinoic acid stimulated macrophages.
  • Useful nucleic acid sequences in this regard include mRNA, genomic DNA and cDNA. For expression, cDNAs are generally preferred because they lack introns that may interfere with expression.
  • a human endothelial cell cDNA library is screened with, e.g., labeled probes from random primed mouse macrophage transglutaminase sequences, which probes preferably span the enzyme's active site and/or putative calcium binding site.
  • an oligo-dT primed cDNA library can be constructed with polyA RNA purified from mouse peritoneal macrophages stimulated with retinoic acid.
  • the library is screened with, e.g., polyclonal antibodies to guinea pig liver tissue transglutaminase and/or labeled RNA probes.
  • Partial clones may be used as probes in additional screening until the complete coding sequence is obtained. If necessary, partial clones are joined in the correct reading frame to construct the complete coding sequence. Joining is achieved by digesting clones with appropriate restriction endonucleases and joining the fragments enzymatically in the proper orientation. Depending on the fragments and the particular restriction endonucleases chosen, it may be necessary to remove unwanted DNA sequences through a "loop out" process of deletion mutagenesis or through a combination of restriction endonuclease cleavage and mutagenesis. It is preferred that the resultant sequence be in the form of a continuous open reading frame, that is, that it lack intervening sequences (introns) .
  • TGHZ3 The sequence of one exemplary mouse clone described herein, TGHZ3, includes 29 nucleotides of 5'-untranslated sequence and 1,775 nucleotides of coding sequence and is shown in Fig. 3 (SEQ. ID. NO. 3).
  • a human cDNA transglutaminase clone isolated as described herein includes the entire 5*-untranslated sequence, as determined by primer extension analysis, the coding domain, and 1,058 nucleotides of 3•-untranslated sequence, as shown in Fig. 3 (SEQ. ID. NO. 1) .
  • This clone lacks a consensus polyadenylation sequence and is slightly shorter than the 3.6 kb full length transcript, as determined by Northern blot analysis of human endothelial cell RNA, suggesting that it lacks approximately 300 bp of 3*-untranslated sequence.
  • the identity of the human tissue transglutaminase clone is confirmed by, for example, in vitro translation.
  • clone hTG-1 encodes a polypeptide that migrates at Mr 80,000 on SDS-polyacrylamide gels. Its deduced molecular weight is 77,253. The active site Cys residue was determined to be at position 277 as shown in Fig. 3.
  • genomic or cDNA sequences encoding tissue transglutaminase may be obtained from libraries prepared from other cells and tissues according to known procedures. For instance, using oligonucleotide probes derived from human endothelial transglutaminase sequences, generally of at least about fourteen nucleotides and up to twenty-five or more nucleotides in length, DNA sequences encoding transglutaminase of other tissues and/or mammalian species may be obtained. If partial clones are obtained, it is necessary to join them in proper reading frame to produce a full length clone, using such techniques as endonuclease cleavage, ligation and loopout mutagenesis.
  • a DNA sequence encoding tissue transglutaminase is inserted into a suitable expression vector, which in turn is used to transform or transfect appropriate host cells for expression.
  • Expression vectors for use in carrying out the present invention will comprise a promoter capable of directing the transcription of a cloned DNA and a transcriptional terminator, operably linked with the sequence encoding the tissue transglutaminase so as to produce a continuously transcribable gene sequence which produces sequences in reading frame and continuously translated to produce a transglutaminase polypeptide.
  • Host cells for use in practicing the present invention include mammalian, avian, plant, insect, bacterial and fungal cells, but preferably eukaryotic cells.
  • Preferred eukaryotic cells include cultured mammalian cell lines (e.g., rodent or human cell lines) and fungal cells, including species of yeast (e.g., Saccharomyces spp., particularly S ⁇ . cerevisiae. Schizosaccharomyces spp., or Kluyveromyces spp.) or filamentous fungi (e.g., Aspergillus spp., Neurospora spp.).
  • yeast e.g., Saccharomyces spp., particularly S ⁇ . cerevisiae. Schizosaccharomyces spp., or Kluyveromyces spp.
  • filamentous fungi e.g., Aspergillus spp., Neurospora spp.
  • Suitable yeast vectors for use in the present invention include YRp7 (Struhl et al., Proc. Natl. Acad. Sci. USA 76: 1035-1039, 1978), YEpl3 (Broach et al.. Gene 8: 121-133, 1979), POT vectors (Kawasaki et al, U.S. Patent No. 4,931,373, which is incorporated by reference herein), pJDB249 and pJDB219 (Beggs, Nature 275:104-108, 1978) and derivatives thereof.
  • Such vectors will generally include a selectable marker, which may be one of any number of genes that exhibit a dominant phenotype for which a phenotypic assay exists to enable transformants to be selected.
  • selectable markers are those that complement host cell auxotrophy, provide antibiotic resistance or enable a cell to utilize specific carbon sources, and include LEU2 (Broach et al., ibid.), UR 3 (Botstein et al., Gene 8: 17, 1979), HIS3 (Struhl et al., ibid.) or P0T1 (Kawasaki et al., ibid.).
  • Another suitable selectable marker is the CAT gene, which confers chloramphenicol resistance on yeast cells.
  • promoters for use in yeast include promoters from yeast glycolytic genes (Hitzeman et al., _s. Biol. Chem. 255: 12073-12080, 1980; Alber and Kawasaki, J. Mol. APPI. Genet. 1: 419-434, 1982; Kawasaki, U.S. Patent No. 4,599,311) or alcohol dehydrogenase genes (Young et al., in Genetic Engineering of Microorganisms for Chemicals. Hollaender et al., (eds.), p. 355, Plenum, New York, 1982; Ammerer, Meth. Enzvmol. 101: 192-201, 1983).
  • particularly preferred promoters are the TPI1 promoter (Kawasaki, U.S. Patent No. 4,599,311, 1986) and the ADH2-4 C promoter (Russell et al.. Nature 304: 652-654, 1983; Irani and Kilgore, U.S. Patent Application Serial No. 183,130, which is incorporated herein by reference) .
  • the expression units may also include a transcriptional terminator.
  • a preferred transcriptional terminator is the TPI1 terminator (Alber and Kawasaki, ibid.).
  • the transglutaminases of the invention may be expressed in Aspergillus spp. (McKnight and Upshall, described in U.S. Patent 4,935,349, which is incorporated herein by reference) .
  • Useful promoters include those derived from Aspergillus nidulans glycolytic genes, such as the ADH3 promoter (McKnight et al., EMBO J. 4:2093-2099, 1985) and the tpiA promoter.
  • An example of a suitable terminator is the ADH3 terminator (McKnight et al., ibid.).
  • cultured mammalian cells may be used as host cells within the present invention.
  • Preferred cultured mammalian cells for use in the present invention include the COS-1 (ATCC CRL 1650) and BALB/c 3T3 (ATCC CRL 163) cell lines.
  • a number of other mammalian cell lines may be used within the present invention, including BHK (ATCC CRL 10314), 293 (ATCC CRL 1573), Rat Hep I (ATCC CRL 1600), Rat Hep II (ATCC CRL 1548), TCMK (ATCC CCL 139), Human lung (ATCC CCL 75.1), Human hepatoma (ATCC HTB-52) , Hep G2 (ATCC HB 8065), Mouse liver (ATCC CCL 29.1), NCTC 1469 (ATCC CCL 9.1) and DUKX cells (Urlaub and Chasin, Proc. Natl. Acad. Sci USA 77: 4216-4220, 1980).
  • Mammalian expression vectors for use in carrying out the present invention will include a promoter capable of directing the transcription of a cloned gene or cDNA.
  • Preferred promoters include viral promoters and cellular promoters.
  • Viral promoters include the immediate early cytomegalovirus promoter (Boshart et al., Cell 41: 521-530, 1985), the SV40 promoter (Subramani et al., Mol. Cell. Biol. 1: 854-864, 1981), and the major late promoter from Adenovirus 2 (Kaufman and Sharp, Mol. Cell. Biol. 2: 1304-1319, 1982).
  • Cellular promoters include the mouse metallothionein-1 promoter (Palmiter et al., U.S. Patent No. 4,579,821), a mouse V promoter (Bergman et al., Proc. Natl. Acad. Sci. USA 81: 7041-7045, 1983; Grant et al., Nuc. Acids Res. 15: 5496, 1987) and a mouse V favor promoter (Loh et al.. Cell 33: 85-93, 1983). Also contained in the expression vectors is a polyadenylation signal located downstream of the coding sequence of interest.
  • a polyadenylation signal located downstream of the coding sequence of interest.
  • Polyadenylation signals include the early or late polyadenylation signals from SV40 (Kaufman and Sharp, ibid.), the polyadenylation signal from the Adenovirus 5 E1B region and the human growth hormone gene terminator (DeNoto et al., Nuc. Acids Res. 9: 3719-3730, 1981).
  • Vectors can also include enhancer sequences, such as the SV40 enhancer and the mouse ⁇ enhancer (Gillies, Cell 33: 717-728, 1983).
  • Expression vectors may also include sequences encoding the adenovirus VA RNAs. Vectors can be obtained from commercial sources (e.g., Stratagene, La Jolla, CA) .
  • Cloned DNA sequences may be introduced into cultured mammalian cells by, for example, calcium phosphate-mediated transfection (Wigler et al., Cell 14: 725, 1978; Corsaro and Pearson, Somatic Cell Genetics 7: 603, 1981; Graham and Van der Eb, Virology 52: 456, 1973), electroporation (Neumann et al., EMBO J. 1: 841-845, 1982), or DEAE-dextran mediated transfection (Ausubel et al., (ed.) Current Protocols in Molecular Biology. John Wiley and Sons, Inc., NY (1987), incorporated herein by reference) .
  • a selectable marker is generally introduced into the cells along with the gene or cDNA of interest.
  • Preferred selectable markers for use in cultured mammalian cells include genes that confer resistance to drugs, such as neomycin, hygromycin, and methotrexate.
  • the selectable marker may be an amplifiable selectable marker.
  • Preferred amplifiable selectable markers are the DHFR gene and the neomycin resistance gene. Selectable markers are reviewed by Thilly (Mammalian Cell Technology. Butterworth Publishers, Stoneham, MA, which is incorporated herein by reference) . The choice of selectable markers is well within the level of ordinary skill in the art.
  • Selectable markers may be introduced into the cell on a separate vector at the same time as the transglutaminase sequence of interest, or they may be introduced on the same vector. If on the same vector, the selectable marker and the transglutaminase sequence of interest may be under the control of different promoters or the same promoter, the latter arrangement producing a dicistronic message. Constructs of this type are known in the art (for example, Levinson and Simonsen, U.S. Patent No. 4,713,339). It may also be advantageous to add additional DNA, known as "carrier DNA" to the mixture which is introduced into the cells.
  • carrier DNA additional DNA
  • Transfected mammalian cells are allowed to grow for a period of time, typically 1-2 days, to begin expressing the DNA sequence(s) of interest. Drug selection is then applied to select for growth of cells that are expressing the selectable marker in a stable fashion. For cells that have been transfected with an amplifiable selectable marker the drug concentration may be increased in a stepwise manner to select for increased copy number of the cloned sequences, thereby increasing expression levels.
  • Promoters, terminators and methods for introducing expression vectors encoding transglutaminase into plant, avian and insect cells are well known in the art.
  • the use of baculoviruses, for example, as vectors for expressing heterologous DNA sequences in insect cells has been reviewed by Atkinson et al. (Pestic. Sci. 28: 215-224,1990).
  • the use of Agrobacterium rhizogenes as vectors for expressing genes in plant cells has been reviewed by Sinkar et al. (J . Biosci. (Banglaore. 11: 47-58, 1987).
  • Host cells containing DNA constructs of the present invention are then cultured to produce the transglutaminase.
  • the cells are cultured according to standard methods in a culture medium containing nutrients required for growth of the chosen host cells.
  • suitable media are known in the art and generally include a carbon source, a nitrogen source, essential amino acids, vitamins and minerals, as well as other components, e.g., growth factors or serum, that may be required by the particular host cells.
  • the growth medium will generally select for cells containing the DNA construct by, for example, drug selection or deficiency in an essential nutrient which is complemented by the selectable marker on the DNA construct or co-transfe ⁇ ted with the DNA construct.
  • Yeast cells are preferably cultured in a medium which comprises a nitrogen source (e.g., yeast extract) , inorganic salts, vitamins and trace elements.
  • the pH of the medium is preferably maintained at a pH greater than 2 and less than 8, preferably at pH 5-6.
  • Methods for maintaining a stable pH include buffering and constant pH control, preferably through the addition of sodium hydroxide.
  • Preferred buffering agents include succinic acid and Bis-Tris (Sigma Chemical Co., St. Louis, MO).
  • Cultured mammalian cells are generally cultured in commercially available serum-containing or serum-free media. Selection of a medium appropriate for the particular cell line used is within the level of ordinary skill in the art.
  • human tissue transglutaminase is expressed in yeast as an intracellular product.
  • the yeast host is a diploid strain homozygous for pep4. a mutation that reduces vacuolar protease levels, as described in Jones et al., Genetics 85:23-33 (1977), incorporated herein by reference.
  • the strain is also homozygous for disruption of the endogenous TPI (triose phosphate isomerase) gene, thereby allowing the S___ pombe P0 1 gene to be used as a selectable marker.
  • the vector includes the P0T1 marker, a leu2-d marker and the ADH2-4c promoter. The P0T1 marker in the TPI " host allows for selection by growth in glucose.
  • the host strain is grown in glucose-containing synthetic media with a glucose feed. An ethanol feed is then substituted for glucose to de-repress the promoter. The pH is maintained with NaOH.
  • Other preferred means for expression are generally described in, e.g., EPO publication EP 268,772, incorporated herein by reference.
  • tissue transglutaminase produced according to the present invention may be purified by affinity chro atography on an antibody column using antibodies directed against transglutaminase. Additional purification may be achieved by conventional chemical purification means, such as liquid chromatography, gradient centrifugation, and gel electrophoresis, among others. Methods of protein purification are known in the art (see generally, Scopes, R. , Protein Purification . Springer-Verlag, NY (1982), which is incorporated herein by reference) and may be applied to the purification of the recombinant transglutaminase described herein.
  • Substantially pure recombinant tissue transglutaminase of at least about 50% is preferred, at least about 70-80% more preferred, and 95-99% or more homogeneity most preferred, particularly for pharmaceutical uses.
  • the recombinant tissue transglutaminase may then be used in food preparation, protein chemistry, therapeutically, etc.
  • tissue transglutaminases may be used in the preparation of food material, such as paste food, cheese, and can be added to dehydrated fish to prevent deterioration caused by protozoans, e.g., myxamoeba.
  • the transglutaminases can also be used in the preparation of ground meat of okiomi (Euphasia superba) , by adding to dehydrated meat parts from 0.1 to 100 units, preferably about 1-40 U per gram of protein to improve meat texture and quality.
  • Frozen granular meats can be improved by combining meat material with tissue transglutaminase of the invention at 1-500 U per gram protein, at 30-60 'C for 10-120 min. to promote crosslinking between gluta ine groups and lysine contained in meat preparations.
  • tissue transglutaminases described herein include the enzyme-catalyzed labeling of proteins and cell membranes (Iwanij, Eur. J. Biochem. 80:359-368 (1977), incorporated herein by reference) , in the introduction of cleavable crosslinks, and in the solid phase reversible removal of specific proteins from biological systems.
  • the human transglutaminase of the invention also can be used therapeutically in humans.
  • the transglutaminase may be used in the repair of wounds and ulcerated lesions.
  • the tissue enzyme is relatively stable, active extracellularly, and binds avidly to collagen, it can be used to stabilize basement membrane structures.
  • An appropriate endogenous substrate for the enzyme is fibronectin, which thus serves as a basis for crosslinking and stabilizing collagen/fibronectin complexes.
  • Transglutaminase expression can be used as a marker for screening for agonists and antagonists of cellular apoptosis.
  • tissue transglutaminase or the nucleic acids which encode the tissue transglutaminase of the invention can also be used to identify agents which induce apoptotic activity by a cell, for the control of, e.g., hyperproliferative disorders.
  • the growth of cells such as adipocytes can be regulated with agents identified using the tissue transglutaminases provided herein as a marker, providing a means for controlling fat depots in certain forms of obesity without the necessity for surgical intervention.
  • Sequences which encodes transglutaminases may be directly detected in cells with labeled synthetic oligonucleotide probes in a hybridization procedure similar to the Southern or dot blot.
  • the polymerase chain reaction (Saiki et al.. Science 239:487 (1988), and U.S. Pat. No. 4,683,195) may be used to amplify DNA sequences, which are subsequently detected by their characteristic size on agarose gels, Southern blot of the gels using transglutaminase sequences or a oligonucleotide probe, or a dot blot using similar probes.
  • the probes may comprise from about 14 nucleotides to about 25 or more nucleotides, sometimes 40 to 60 nucleotides, and in some instances a substantial portion or even the entire cDNA of a transglutaminase gene of the invention may be used.
  • the probes are labeled with a detectable signal, such as an enzyme, biotin, a radionuclide, fluorophore, chemiluminescer, paramagnetic particle, etc.
  • a detectable signal such as an enzyme, biotin, a radionuclide, fluorophore, chemiluminescer, paramagnetic particle, etc.
  • mice (1200) were sacrificed and their peritoneal cavities were washed with RPMI. Cells were harvested from the wash by centrifugation. The cells were plated and macrophages were allowed to attach to the dishes for 60 minutes.
  • the dishes were then washed and the macrophages were recovered.
  • the macrophages were stimulated with retinoic acid (10 ⁇ 6 M) for 6 hours.
  • RNA was then isolated from the cells, cDNA was synthesized and E. coli cells were infected with recombinant phages.
  • RNA derived from control and retinoic acid stimulated mouse macrophages was used to locate the transcription start site.
  • a synthetic oligonucleotide of sequences included in the 5' end of cDNA clone TG7.4 was hybridized to macrophage RNA and then transcripts were synthesized in the presence of P-dATP.
  • a single prominent 255 nucleotide band, more abundant in transcripts from retinoic acid-stimulated than control macrophage RNA was detected, locating the transcription start site at a position 103 nucleotides upstream of the initiation ATG.
  • an oligo-dT and random-primed cDNA library was constructed with polyA + RNA from human umbilical vein endothelial cells (HUVEC) in the vector lambda ZAP (Stratagene Inc., La Jolla, CA) .
  • X- LI blue cells (Stratagene) were infected with recombinant phages and 2 x 10 plagues were screened with a random-primed mouse macrophage transglutaminase [ P]-labeled DNA probe spanning the active site and the putative calcium binding domain (TG7.4) to facilitate isolation of full length cDNA.
  • the hybridization procedure was done generally as described in Ausubel et al., ibid., at 55*C overnight with a final wash at 60*C in 0.1% SSC/0.1% SDS for 30 minutes.
  • the initial screen of 2 x 10 recombinant phage yielded 5 positive clones.
  • the inserts in three of the clones, hTG2, 3 and 5, were totally included within the largest cDNA clone, hTGl, which was approximately 3.3 kilobases.
  • Example 2 describes the sequencing of mouse and human cDNA clones obtained in Example I. The results show a substantial degree of sequence homology between the two species of tissue enzyme.
  • both strands of the human tissue transglutaminase cDNA clone were sequenced by the dideoxy chain termination method with a S& enase enzyme kit using synthetic oligonucleotide primers and deleted clones derived by exonuclease digestion (Fig. 2) .
  • Fig. 3 SEQ. ID. NO. 1
  • the 3257 nucleotides included a single open reading frame encoding 687 amino acids (also SEQ.ID. NO. 2).
  • initiation codon located 136-138 nucleotides downstream from the transcription start site, was included within a consensus sequence (ACCATGG) recognized as optimal for the initiation of eukaryotic translation (Kozak sequence; Kozak, Cell 44:283-292 (1986)).
  • a terminator codon located at nucleotide 2194- 2196, was followed by 1058 nucleotides of 3•-untranslated sequence. No consensus polyadenylation signal sequence was recognized in the 3'-untranslated region.
  • the nucleotide sequence of the mouse tissue transglutaminase (Fig. 3) (SEQ. ID. No. 3) was determined by sequencing of overlapping cDNA clones (Fig. 1) .
  • CsCl purified, double-stranded mouse cDNA was sequenced by both the chem_cal degradation (Maxam and Gilbert, Proc. Natl. Acad. Sci. USA 74:560-564 (1977)) and the dideoxy chain termination (Sanger et al., Proc. Natl. Acad. Sci. USA 74:5463-5467 (1977)) methods.
  • the entire murine transcription unit sequence was slightly larger than the human transglutaminase cDNA clone (Fig. 3) .
  • the initiation ATG was included in a Kozak sequence.
  • the terminator triplet (TAA) was followed by 1400 bp of 3'- untranslated sequence that included a consensus polyadenylation sequence (AATAAA) at the 3' end of the clone.
  • Fig. 3 compares the nucleotide sequences of the human and mouse tissue transglutaminase cDNA's.
  • the sequence of the human enzyme is fully represented in the top line and the deduced amino acid sequence of the coding domain is shown below.
  • the third line contains the deduced amino acid sequence of the mouse tissue transglutaminase. Residues identical with the human enzyme are shown in an asterisk, residues that are distinct are shown with the single letter code.
  • the fourth line shows the nucleotide sequence of the mouse transglutaminase. Nucleotides identical to the human enzyme are shown with a dash.
  • Hybridization probes suitable for detecting tissue transglutaminase mRNA in tissues were prepared as TG1600 antisense RNA ( 32 P labeled using 32 P-UTP) .
  • Northern blots were performed according to Thomas, Proc. Natl. Acad. Sci. USA 77:5201 (1980), incorporated herein by reference.
  • Fig. 4 shows the Northern blot analysis of RNA from human umbilical vein endothelial cells probed with radiolabeled cDNA prepared form the insert in clone hTG-1. A single band at approximately 3.5 kilobases was detected.
  • Fig. 4 shows the Northern blot analysis of RNA from human umbilical vein endothelial cells probed with radiolabeled cDNA prepared form the insert in clone hTG-1. A single band at approximately 3.5 kilobases was detected.
  • RNA's prepared from several mouse tissues (liver, spleen, kidney, testis, heart, lungs, thymus, and brain) .
  • Minimal levels of transglutaminase mRNA were detected in thymus and in brain tissues. The levels of this RNA were higher in liver, spleen and testis and were highest in the kidney, lung and heart.
  • a cDNA clone for human endothelial cell tissue transglutaminase (clone hTGl) was cloned into the Eco RI site of the eukaryotic expression vector pSG5 (Stratagene) .
  • This 3257 bp insert contained 138 nucleotides of 5'-untranslated sequence, the coding region of the enzyme and 1058 bp of 3'- untranslated sequence.
  • the human tissue transglutaminase expression plasmid was transiently transfected into COS-1 cells using a DEAE-Dextran mediated transfection protocol.
  • Cells were cultured in DMEM containing 10% fetal calf serum (FCS) . After 48 and 72 hours cells were washed, scraped and homogenized and the transglutaminase activity was measured as the calcium- dependent covalent incorporation of radiolabeled putrescine into N,N-dimethylcasein (essentially as described by Murtagh et al., J. Biol. Chem. 261:614-621 (1986)).
  • transglutaminase activity was 5.6 fmols/min/mg.
  • the transglutaminase activity was 270 fmols/min/mg.
  • BALB/c 3T3 cells were co-transfected (via the CaP0 4 procedure) with the transglutaminase expression vector and an SV-neo containing plasmid (obtained from Clontech) .
  • the cells were grown for 48 hours in DMEM containing 10% FCS and 10% Serum Plus (Hazelton Biologies, Inc., Lexena, KS) .
  • the cells were washed and the medium was replaced with DMEM containing 10% FCS, 10% Serum Plus and 400 ⁇ g/ l G-418.
  • G-418 resistant cells were cloned. Individual clones of transfected 3T3 cells were grown to confluency.
  • tissue transglutaminase was measured by Western blot (U.S. Pat. No. 4,452,901; Towbin et al., Proc. Natl. Acad. Sci. USA 76:4350-4358 (1979)) using a polyclonal antibody to guinea pig liver tissue transglutaminase and by enzymatic assay (using the assay described above) .
  • Three clones of 3T3 cells (clones 13, 15, and 19) were isolated and characterized in detail.
  • GCCCCCGCCC CGACC ATG GCC GAG GAG CTG GTC TTA GAG AGG TGT GAT CTG 171
  • ATC CGT GTG GGC CAG AGC ATG AAC ATG GGC AGT GAC TTT GAC GTC TTT 1611 lie Arg Val Gly Gin Ser Met Asn Met Gly Ser Asp Phe Asp Val Phe 480 485 490
  • GGC CCC GCC TAA GGGACCCCTG CTCCCAGCCT GCTGAGAGCC CCCACCTTGA 2239
  • GCT GCC AAC AGC TAC CTG CTG GCT GAG AGA GAT CTC TAC GTG GAG AAT 1781 Ala Ala Asn Ser Tyr Leu Leu Ala Glu Arg Asp Leu Tyr Val Glu Asn 570 575 580

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Abstract

Des transglutaminases tissulaires humaines et murines sont clonées, séquencées et exprimées. Les transglutaminases tissulaires selon l'invention sont utiles dans, entre autres, la réparation thérapeutique des plaies, la stabilisation de préparations alimentaires, et en tant que marqueurs pour des agents d'identification qui agissent comme agonistes ou antagonistes de l'apoptose cellulaire.
PCT/US1991/009784 1991-01-04 1991-12-30 Clonage et expression de transglutaminases tissulaires WO1992012238A1 (fr)

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Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994010296A1 (fr) * 1992-11-03 1994-05-11 Oklahoma Medical Research Foundation Gene de la transglutaminase
EP0598133A1 (fr) * 1992-04-21 1994-05-25 Ajinomoto Co., Inc. Remede contre les plaies
US5525336A (en) * 1993-02-19 1996-06-11 Green; Howard Cosmetic containing comeocyte proteins and transglutaminase, and method of application
US5736132A (en) * 1993-06-03 1998-04-07 Orthogene, Inc. Method of promoting adhesion between tissue surfaces
WO1999010507A1 (fr) * 1997-08-29 1999-03-04 Wisconsin Alumni Research Foundation Transglutaminase et gene codant cette derniere
US5958752A (en) * 1993-04-30 1999-09-28 The United States Of America As Represented By The Department Of Health And Human Services Nucleic acid molecules encoding human trichohyalin and use thereof
US6190896B1 (en) 1997-11-14 2001-02-20 Bassam M. Fraij Active human cellular transglutaminase
US6267957B1 (en) 1998-01-20 2001-07-31 Howard Green Attaching agents to tissue with transglutaminase and a transglutaminase substrate
WO2001070787A1 (fr) * 2000-03-10 2001-09-27 Shanghai Biowindow Gene Development Inc. Nouveau polypeptide, glutamyl-aminase inverse humaine 12, et polynucleotide codant pour ce polypeptide
US6919076B1 (en) 1998-01-20 2005-07-19 Pericor Science, Inc. Conjugates of agents and transglutaminase substrate linking molecules
US6958148B1 (en) 1998-01-20 2005-10-25 Pericor Science, Inc. Linkage of agents to body tissue using microparticles and transglutaminase
US10005846B2 (en) 2012-05-24 2018-06-26 Lifearc Anti-transglutaminase 2 antibodies

Citations (2)

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EP0268772A2 (fr) * 1986-09-19 1988-06-01 ZymoGenetics, Inc. Expression du facteur XIII biologiquement actif
US4929554A (en) * 1981-10-19 1990-05-29 Goeddel David V Human immune interferon

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US4929554A (en) * 1981-10-19 1990-05-29 Goeddel David V Human immune interferon
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Title
2nd INTERNATIONAL CONFERENCE ON TRANSGLUTAMINASES & PROTEIN CROSS-LINKING REACTIONS, 24-28 June 1990, GENTILE et al., "Molecular Cloning and Sequnce Analysis of cDNa for a Retinoic Acid-Inducible Tissue Transglutaminase from Human Umbilical Vein Endothelial Cells", see abstract. *
FASEB JOURNAL, Vol. 3, No. 4, issued 17 February 1989, SAYDAK et al., "cDNA Cloning of mouse Macrophage Tissue Transglutaminase", page A1209, see abstract 5708. *
THE JOURNAL OF CELL BIOLOGY, Vol. 109, No. 4, Part 2, issued 9 November 1989, GENTILE et al., "Isolation and Characterization of cDNA and Genomic Clones of Human Endothelial Cell Transglutaminases", page 198a, see abstract 1068. *

Cited By (19)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0598133A1 (fr) * 1992-04-21 1994-05-25 Ajinomoto Co., Inc. Remede contre les plaies
EP0598133A4 (fr) * 1992-04-21 1996-03-06 Ajinomoto Kk Remede contre les plaies.
US5525335A (en) * 1992-04-21 1996-06-11 Ajinomoto Co., Inc. Wound healing agent
WO1994010296A1 (fr) * 1992-11-03 1994-05-11 Oklahoma Medical Research Foundation Gene de la transglutaminase
US5726051A (en) * 1992-11-03 1998-03-10 Oklahoma Medical Research Foundation Transglutaminase gene
US5525336A (en) * 1993-02-19 1996-06-11 Green; Howard Cosmetic containing comeocyte proteins and transglutaminase, and method of application
US5958752A (en) * 1993-04-30 1999-09-28 The United States Of America As Represented By The Department Of Health And Human Services Nucleic acid molecules encoding human trichohyalin and use thereof
US5736132A (en) * 1993-06-03 1998-04-07 Orthogene, Inc. Method of promoting adhesion between tissue surfaces
WO1999010507A1 (fr) * 1997-08-29 1999-03-04 Wisconsin Alumni Research Foundation Transglutaminase et gene codant cette derniere
US6020178A (en) * 1997-08-29 2000-02-01 Wisconsin Alumni Research Foundation Transglutaminase and gene encoding same
US6114119A (en) * 1997-08-29 2000-09-05 Wisconsin Alumni Research Foundation Transglutaminase and gene encoding same
US6190896B1 (en) 1997-11-14 2001-02-20 Bassam M. Fraij Active human cellular transglutaminase
US6267957B1 (en) 1998-01-20 2001-07-31 Howard Green Attaching agents to tissue with transglutaminase and a transglutaminase substrate
US6919076B1 (en) 1998-01-20 2005-07-19 Pericor Science, Inc. Conjugates of agents and transglutaminase substrate linking molecules
US6958148B1 (en) 1998-01-20 2005-10-25 Pericor Science, Inc. Linkage of agents to body tissue using microparticles and transglutaminase
WO2001070787A1 (fr) * 2000-03-10 2001-09-27 Shanghai Biowindow Gene Development Inc. Nouveau polypeptide, glutamyl-aminase inverse humaine 12, et polynucleotide codant pour ce polypeptide
US10005846B2 (en) 2012-05-24 2018-06-26 Lifearc Anti-transglutaminase 2 antibodies
US10961319B2 (en) 2012-05-24 2021-03-30 Lifearc Anti-transglutaminase 2 antibodies
US11718686B2 (en) 2012-05-24 2023-08-08 Lifearc Anti-transglutaminase 2 antibodies

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